Methods of treating a meiotic kinesin associated disease

ABSTRACT

The invention provides methods of treating a meiotic kinase-associated disease, preferably the meiotic kinase HSET, by administering an inhibitor of the meiotic kinase. Preferably, the disease is associated with the presence of supernumerary centrosomes, such as cancer. Methods of inhibiting the growth of a tumor cell by contacting the cell with an inhibitor of a meiotic kinase, preferably HSET, are also provided. Screening methods for identifying inhibitors of the meiotic kinase HSET are also provided. Methods of selecting subjects for treatment with an inhibitor of a meiotic kinase, such as HSET, are also provided.

BACKGROUND OF THE INVENTION

Centrosomes play a crucial role in the equal segregation of chromosomesby contributing to bipolar spindle assembly during mitosis (Doxsey, S.(2001) Nat Rev Mol Cell Biol 2:688-698). The tight control on centrosomeduplication, limiting it to once per cell cycle, ensures that normalcells enter mitosis with two centrosomes or microtubule organizingcenters (MTOCs). Failure to properly control centrosome number andfunction can lead to multipolar spindles, aneuploidy, disruption of cellpolarity, and failure of asymmetric cell divisions (Heneen, W. K. (1970)Chromosoma 29:88-117; Nigg, E. A. (2002) Nat Rev Cancer 2:815-525).

Increased centrosome number, often termed centrosome amplification, is acommon characteristic of solid and hematological cancers. Centrosomeamplification correlates with aneuploidy and malignant behavior in tumorcell lines, mouse tumor models, and human tumors (D'Assoro A. B. et al.(2002) Breast Cancer Res Treat 75: 25-34; Giehl S. et al. (2005)Leukemia 19:1192-1197; Levine, D. S. et al. (1991) Proc Natl Acad SciUSA 88:6427-6431; Lingle, W. L. et al. (1998) Proc Natl Acad Sci USA95:2950-2955; Pihan, G. A. et al. (2003) Cancer Res 63:1398-1404).Mutation or misregulation of a variety of tumor suppressors or oncogenesare correlated with centrosome amplification (Fukasawas K. (2007) NatRev Cancer 7:911-924). Centrosome amplification can, in principle, arisefrom several types of cell division errors; centrosome overduplication,de novo synthesis of centrosomes, cell fusion, or cytokinesis failure(Boveri, T. (1929) The Origin of Malignant Tumors (Baltimore; Williamsand Wilkins); Ganem, N. J. et al. (2007) Curr Opin Genet Dev 17:157-162;Nigg, E. A. (2002) Nat Rev Cancer 2:815-825).

The role of supernumerary centrosomes in tumor biology is likely to bemultifaceted. Whereas multiple centrosomes might facilitatetumorigenesis by promoting aneuploidy and/or disrupting cell polarity,they may also impose a fitness cost on the growth of mature cancersbecause of the potential for multipolar mitoses. To circumvent thisproblem, many cancer cells appear to have mechanisms that suppressmultipolar mitoses, the best studied being clustering of supernumerarycentrosomes into two groups enabling a bipolar mitosis (Brinkley, B. R.(2001) Trends Cell Biol 11:18-21; Nigg, E. A. (2002) Nat Rev Cancer2:815-825; Ring, D. et al. (1982) J Cell Biol 94:549-556).

Centrosome clustering in tumor cells is incompletely understood,however, it is expected to rely to a significant degree onMicrotubule-associated proteins (MAPs) and motors that organize thespindle poles (Karsenti, E. and Vernos, I. (2001) Science 294:543-547,Nigg, E. A. (2002) Nat Rev Cancer 2:815-825) For example, recent workuncovered a requirement of cytoplasmic dynein, a minus end-directedmicrotubule (MT) motor, and NuMA, a spindle associated MAP, incentrosome clustering (Quintyne, N. J. et al. (2005) Science307:127-129). The existence of mechanisms that suppress multipolarmitoses raises the possibility of a novel therapeutic strategy forcancer: drugs that interfere with centrosome clustering mechanisms couldbe lethal to tumor cells containing multiple centrosomes, butpotentially spare normal cells. Although several drugs, including Taxol,can promote multipolar mitosis, none are specific for cells withmultiple centrosomes (Chen, J. G. and Horwitz, S. B. (2002) Cancer Res62:1935-1938 Rebacz, B. et al. (2007) Cancer Res 77:6342-6350).

Accordingly, identification of components involved in the centrosomeclustering mechanisms in tumor cells is still needed.

SUMMARY OF THE INVENTION

The present invention identifies a key component involved in thecentrosome clustering mechanism in tumor cells and demonstrates thatcentrosome declustering, by inhibition of this component, can inducecell death selectively in cells with supernumerary centrosomes. This keycomponent, the meiotic kinesin HSET (a kinesin-14 family member), is notessential for mitosis in normal cells but is demonstrated herein to beessential for the survival of cancer cells with extra centrosomes.Accordingly, the present invention provides a target for selectivekilling of cells containing extra centrosomes, such as cancer cells,while avoiding killing of normal cells.

Thus, in one aspect, the invention pertains to a method of treating ameiotic kinesin-associated disease or disorder, comprising administeringto a subject in need of treatment thereof an agent which inhibits ameiotic kinesin such that treatment of the disease or disorder isachieved. In one embodiment, the disease or disorder is an autosomaldisease or disorder. In a preferred embodiment, the disease or disorderis a centrosomal disease or disorder (e.g., characterized by thepresence of supernumerary centrosomes). In another preferred embodiment,the disease or disorder is a cellular proliferative disease, such ascancer or malignancy. Preferably, the meiotic kinesin is a member of thekinesin-14 family, most preferably HSET. Examples of suitable agents forinhibiting the meiotic kinesin include RNAi agents and small molecules.In one embodiment, the agent inhibits the ATPase activity of thekinesin. In another embodiment, the agent inhibits the microtubulebinding activity of the kinesin. The agent can be administered, forexample, orally or parentally.

In another aspect, the invention pertains to a method of inhibitinggrowth of a tumor cell in which cellular proliferation is associatedwith a meiotic kinesin, comprising contacting the tumor cell with anagent which inhibits a meiotic kinesin such that growth of the tumorcell is inhibited. In a preferred embodiment, the tumor cell comprisessupernumerary centrosomes. Preferably, the meiotic kinesin is a memberof the kinesin-14 family, most preferably HSET. Examples of suitableagents for inhibiting the meiotic kinesin include RNAi agents and smallmolecules. In one embodiment, the agent inhibits the ATPase activity ofthe kinesin. In another embodiment, the agent inhibits the microtubulebinding activity of the kinesin. The tumor cell can be contacted withthe agent by, for example, culturing the tumor cell with the agent or bydirectly injecting the agent into a tumor that contains the tumor cellor by administering the agent to a subject bearing a tumor that containsthe tumor cell.

In another aspect, the invention pertains to a method of inhibitingproliferation of a cell in which cellular proliferation is associatedwith a meiotic kinesin, comprising contacting the cell with an agentwhich inhibits a meiotic kinesin such that inhibition of cellproliferation is achieved. In a preferred embodiment, the cell containssupernumerary centrosomes. Preferably, the meiotic kinesin is a memberof the kinesin-14 family, most preferably HSET. Examples of suitableagents for inhibiting the meiotic kinesin include RNAi agents and smallmolecules. In one embodiment, the agent inhibits the ATPase activity ofthe kinesin. In another embodiment, the agent inhibits the microtubulebinding activity of the kinesin.

In yet another aspect, the invention pertains to a method foridentifying a compound that inhibits activity of a meiotic kinesin HSET,the method comprising

providing an indicator composition comprising the meiotic kinesin HSET;

contacting the indicator composition with a test compound; and

determining activity of the meiotic kinesin HSET in the presence of thetest compound, wherein reduction of activity of the meiotic kinesin HSETin the presence of the test compound, as compared to activity of themeiotic kinesin HSET in the absence of the test compound, identifies thetest compound as a compound that inhibits the activity of a meiotickinesin HSET.

In a preferred embodiment, the indicator composition is contacted witheach member of a library of test compounds and one or more testcompounds within the library are selected that inhibit the activity ofthe meiotic kinesin HSET.

In one embodiment, the activity of the meiotic kinesin HSET isdetermined by measuring the ATPase activity of the kinesin. In anotherembodiment, the activity of the meiotic kinesin HSET is determined bymeasuring the microtubule binding activity of the kinesin. In yetanother embodiment, the activity of the meiotic kinesin HSET isdetermined by measuring the expression of HSET mRNA or protein.

The invention also pertains to isolated compounds identified by thescreening methods of the invention.

In yet another aspect, the invention pertains to a method of selecting asubject with a tumor or for treatment with an agent which inhibits ameiotic kinesin, the method comprising (i) obtaining as tumor cellsample from the subject, and (ii) determining centrosome number in thetumor cell sample, wherein presence of supernumerary centrosomes in thetumor cell sample selects the subject for treatment with an agent whichinhibits a meiotic kinesin. Preferably, the meiotic kinesin is a memberof the kinesin-14 family, most preferably HSET. Preferably, at least 50%of the tumor cells in the sample contain supernumerary centrosomes, morepreferably at least 75% of the tumor cells in the sample containsupernumerary centrosomes and even more preferably at least 90% of thetumor cells in the sample contain supernumerary centrosomes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the scheme for the genome-wide RNAi screen in Drosophila S2cells to identify genes whose knockdown leads to multipolar spindles(centrosome de-clustering).

FIG. 2 is a bar graph of the percentage of S2 cells with abnormalspindles upon RNAi knockdown of EGFP or Mad2 alone, or RNAi knockdown ofEFGP or Mad2 plus treatment with MG132.

FIG. 3 is a bar graph of the percentage of S2 cells with abnormalspindles upon treatment with Latrunculin (LatA), Cytochalasin D orConcanavalin-A (Con-A).

FIG. 4 is a bar graph of the percentage of cells with multipolarspindles in MCF-7. MDA-231, MMECS 4N or NIE-115 cells upon treatmentwith DMSO, LatA or DCB

FIG. 5 is a Western blot showing the depletion of HSET after three daysof siRNA in MDA-231 and MCF-7 cells.

FIG. 6 is a Western blot showing the depletion of Myo10 after three daysof siRNA in MDA-231 and MCF-7 cells.

FIG. 7 is a bar graph showing the percentage of cells with multipolarspindles in MCF-7 or MDA-231 cells upon treatment with siRNA of HSET orMyo10.

FIG. 8 is a bar graph showing the percentage of mitotic cells withmultipolar spindles upon treatment with siRNA of HSET or Myo10, bothwith (+) or without (−) LatA.

FIG. 9 is bar graphs showing the loss of cell viability and inhibitionof colony formation by NIE-115 cells after 6 days HSET siRNA treatment.

FIG. 10 is a bar graph showing HSET RNAi-induced cell death in variouscaner cell lines in proportion to the fraction of cells with extracentrosomes.

DETAILED DESCRIPTION

The present invention identifies a key component involved in thecentrosome clustering mechanism in tumor cells. The present inventionfurther demonstrates that centrosome declustering can induce cell deathselectively in cells with supernumerary centrosomes. In at least oneembodiment, the present invention is based, at least in part, on thediscovery that the meiotic kinesin HSET, a normally nonessential kinesinmotor, is required for the viability of cells containing extracentrosomes. Multiple centrosomes in tumor cells create the potentialfor multipolar divisions that can lead to aneuploidy and cell death.Nevertheless, many cancer cells successfully divide because ofmechanisms that suppress multipolar mitoses. A genome-wide RNAi screenin Drosophila S2 cells and a secondary analysis in cancer cells definedmechanisms that suppress multipolar mitoses. In particular, the meiotickinesin HSET now has been shown to be essential for the viability ofcertain extra centrosome-containing cancer cells, with inhibition ofHSET leading to inhibition of cell viability (see in particular Examples6 and 8 herein). Thus, the finding described herein that the minusend-directed motor HSET is essential for clustering extra centrosomesprovides a new therapeutic strategy: blocking centrosome clustering andpromoting multipolar mitoses to selectively induce death in tumors witha high proportion of cells containing multiple centrosomes. The presentinvention provides for assays to identify agents that modulate theactivity of a meiotic kinesin, such as HSET, as well as methods oftreating diseases or disorders associated with meiotic kinesin activity,and methods of selecting subjects for treatment with a meiotic kinesininhibitor.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “kinesin” refers to a class of motor proteins found ineukaryotic cells that are capable of moving along microtubules poweredby the hydrolysis of ATP. The term “meiotic kinesin” refers to kinesinsthat are involved in, and necessary for, the cellular function ofmeiosis.

The term “kinesin-14 family member” refers to a kinesin that a member ofthe kinesin-14 family, which family shares a common C-terminal motordomain differing from other kinesin proteins. The kinesin-14 family isalso known in the art as the C-terminal kinesins, examples of kinesin-14family members include Homo sapiens proteins HSET, CHO2, KIFC2 andKIFC3, Mus musculus proteins HSET and KIFC2, Drosophila melanogasterprotein Ncd and Saccharomyces cerevisiae protein Kar3.

The term “HSET” refers to a kinesin-14 family member also known in theart as KIFC1 (kinesin family member C1). The mRNA and protein sequenceof human HSET are available at Genbank Accession Nos. NM_(—)002243 andNP_(—)002254, respectively. The mRNA and protein sequence of mouse HSETare available at Genbank Accession Nos. NM_(—)053173 and NP_(—)444403,respectively. A human HSET sequence may differ from human HSET ofGenbank Accession Number NP_(—)002254 by having, for example, conservedmutations or mutations in non-conserved regions and the HSET hassubstantially the same biological function as the human HSET of GenbankAccession Number NP_(—)002254. A particular human HSET sequence willgenerally be at least 90% identical in amino acids sequence to humanHSET, such as to Genbank Accession Number NP_(—)002254, and containsamino acid residues that identify the amino acid sequence as being humanwhen compared to HSET amino acid sequences of other species (e.g.,murine). In certain cases, a human HSET may be at least 95%, or even atleast 96%, 97%, 98%, or 99% identical in amino acid sequence to humanHSET, such as to Genbank Accession Number NP_(—)002254. In certainembodiments, a human HSET sequence will display no more than 10 aminoacid differences from the human HSET sequence, such as to GenbankAccession Number NP_(—)002254. In certain embodiments, the human HSETmay display no More than 5, or even no more than 4, 3, 2, or 1 aminoacid difference from the human HSET sequence, such as to GenbankAccession Number NP_(—)002254.

The term “meiotic kinesin-associated disease or disorder” is intended torefer to a disease or disorder the pathogenesis of which requires theactivity of a kinesin known to function in meiosis.

The term “autosomal disease or disorder” is intended to refer to adisease or disorder the pathogenesis of which is associated with anon-sex chromosome.

The term “centrosomal disease or disorder” is intended to refer to adisease or disorder the pathogenesis of which is associated withalteration in the number or activity of centrosomes.

The term “supernumerary centrosomes” is intended to refer to more thanthe usual or prescribed number of centrosomes in a cell, typically morethan the usual number of two centrosomes in a cell.

The term “cellular proliferative disease or disorder” is intended torefer to a disease or disorder the pathogenesis of which is associatedwith altered or aberrant cellular proliferation.

As used herein, an agent or compound that “inhibits activity” of ameiotic kinesin, such as HSET, is intended to refer to an agent orcompound that reduces, or decreases, or lessens the activity of themeiotic kinesin, which inhibition can be partial or complete, andwherein such “activity” can be, for example, expression of the mRNAencoding the kinesin in a cell, expression of the protein level of thekinesin in a cell, or expression of the enzymatic activity or otherbiological activities of the kinesin, examples of such enzymatic andother biological activities including, but not being limited to, ATPaseactivity and microtubule binding activity.

The term “agent” includes any substance, molecule, element, compound,entity, or a combination thereof. It includes, but is not limited to,e.g., protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, and the like. It can be a natural product, a syntheticcompound, or a chemical compound, or a combination of two or moresubstances. Unless otherwise specified, the terms “agent”, “substance”,and “compound” can be used interchangeably.

As used herein, an “antisense” nucleic acid comprises a nucleotidesequence that is complementary to a “sense” nucleic acid encoding aprotein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, complementary to an mRNA sequence or complementary to thecoding strand of a gene. Accordingly, an antisense nucleic acid canhydrogen bond to a sense nucleic acid.

As used herein, an “RNAi agent” refers to an agent, such as a nucleicacid molecule, that mediates RNA interference. RNA interference (RNAi)is a post-transcriptional, targeted gene-silencing technique that usesdouble-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containingthe same sequence as the dsRNA (see e.g., Sharp, P. A. and Zamore, P. D.(2000) Science 287:2431-2432; Zamore, P. O., et al, (2000) Cell19125-33; Tuschl, T. et al. (1999) Genes Dev. 13:3191-3197; Cottrell T.R., and Doering T. L. (2003) Trends Microbiol. 11:37-43; Bushman F.(2003) Mol Therapy 7:9-10; ‘McManus M. T. and Sharp P. A. (2002) Nat RevGenet. 3:737-47). The process occurs when an endogenous ribonucleasecleaves the longer dsRNA into shorter, e.g., 21- or 22-nucleotide-longRNAs, termed small interfering RNAs or siRNAs. The smaller RNA segmentsthen mediate the degradation of the target mRNA. Kits for synthesis ofsiRNA are commercially available, e.g. from New England Biolabs,Dharmacon or Ambion. Thus, in one embodiment, an “RNAi agent” is anucleic acid molecule that is an siRNA molecule. Other examples ofnucleic acid molecules that can be used to silence gene expression viaRNA interference include small hairpin RNA (shRNA) and microRNA (miRNA).Accordingly, the term “RNAi agent” also is intended to encompassmolecules such as shRNA, miRNA and the like that can be used to silencegene expression by RNA interference.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and no-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

Methods of Treating Diseases or Disorders

In one aspect, the invention pertains to methods for treating diseasesor disorders. In particular, the invention provides a method of treatinga meiotic kinesin-associated disease or disorder. The method comprisesadministering to a subject in need of treatment thereof an agent whichinhibits a meiotic kinesin such that treatment of the disease ordisorder is achieved.

Preferably, the disease or disorder is an autosomal disease or disorder.More preferably, the disease or disorder is a centrosomal disease ordisorder. Even more preferably, the disease or disorder is a cellularproliferative disease or disorder. Most preferably, the disease ordisorder is a cancer, tumor or other malignancy. Preferably, the cancer,tumor or other malignancy is one in which the cancer, tumor or malignantcells comprise supernumerary centrosomes. More preferably, at least 50%of the cancer, tumor or malignant cells comprise supernumerarycentrosomes, even more preferably at least 75% of the cancer, tumor ormalignant cells comprise supernumerary centrosomes, and even morepreferably at least 90% of the cancer, tumor or malignant cells comprisesupernumerary centrosomes. Non-limiting examples of cancers which can betreated according to the methods of the invention include breast, colon,lung, prostate, ovarian, pancreatic, brain, stomach, renal, hepatic,bone, skirt, leukemias, lymphomas, multiple myeloma and melanoma.

Other diseases or disorders associated with the accumulation ofsupernumerary centrosomes include human papillomavirus (HPV) infection,including HPV-associated cervical neoplasias (see e.g., Duensing, S. andMunger, K. (2002) Oncogene 21:6241-6248).

Preferably, the meiotic kinesin is a kinesin-14 family member. Morepreferably, the meiotic kinesin is HSET.

Any agent that inhibits the activity of the meiotic kinesin, e.g., HSET,and that is suitable for use in the subject can be used in the treatmentmethods of the invention. In a preferred embodiment, the agent is anRNAi agent, such as an siRNA. As described in detail in Examples 6 and8, siRNA inhibitors of human HSET were demonstrated to reduce viabilityof several different cancer cell lines. Nonlimiting examples of nucleicacid molecules that can function as siRNA inhibitors of human HSETinclude the oligonucleotides shown in SEQ ID NOs: 1-4. Other agents thatcan be used to inhibit the activity of a meiotic kinesin, e.g., HSET,include, but are not limited to, antisense molecules and small moleculeinhibitors (e.g., small organic molecules). Such agents can beidentified, for example, using screening assays for HSET inhibitors asdescribed in detail herein. In one embodiment, the agent inhibits theATPase activity of the kinesin. In another embodiment, the agentinhibits the microtubule binding activity of the kinesin.

An agent of the invention typically is administered to the subject in apharmaceutical composition. Administration is by any route suitable toaccomplish the desired treatment. For example, in one embodiment, theagent is administered orally. In another embodiment, the agent isadministered parenterally.

The pharmaceutical composition typically includes the agent formulatedtogether with a pharmaceutically acceptable carrier. Pharmaceuticalcompositions can be administered in combination therapy, i.e., combinedwith other agents. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. Preferably, the carrier issuitable for oral, intravenous, intramuscular, subcutaneous, parenteral,spinal or epidermal administration (e.g., by injection on infusion).Depending on the route of administration, the active agent may be coatedin a material to protect the agent from the action of acids and othernatural conditions that racy inactivate the agent.

The pharmaceutical composition may include one or more pharmaceuticallyacceptable salts. A “pharmaceutically acceptable salt” refers to a saltthat retains the desired biological activity of the parent compound anddoes not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such saltsinclude acid addition salts and base addition salts. Acid addition saltsinclude those derived from nontoxic inorganic acids, such ashydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic adds, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition also may include a pharmaceuticallyacceptable anti-oxidant. Examples of pharmaceutically acceptableantioxidants include: (1) water soluble antioxidants, such ascorbicacid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,sodium sulfite and the like; (2) oil-soluble antioxidants, such asascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and (3) metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents that delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthis disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of this disclosure are dictated by anddirectly dependent on (a) the unique characteristics of the activecompound and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present disclosureemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A pharmaceutical composition can be administered via one or more routesof administration using one or more of a variety of methods known in theart. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results.Preferred routes of administration include oral, intravenous,intramuscular, intradermal, intraperitoneal, subcutaneous, spinal orother parenteral routes of administration, for example by injection orinfusion. The phrase “parenteral administration” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular; intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, a therapeutic composition can be administeredwith a needleless hypodermic injection device, such as the devicesdisclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413;4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants andmodules useful in the present disclosure include: U.S. Pat. No.4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicants throughthe skin; U.S. Pat. No. 4,447,2233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196 which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the pharmaceutical compositions can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic agent crosses the BBB (if desired), it canbe formulated, for example, in liposomes. For methods of manufacturingliposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.Parmacol. 29:685). Exemplary targeting moieties include rotate or biotin(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawaet al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995)Antimicrob. Agents Chemother. 9:180); surfactant protein A receptor(Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al.(1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Lankkanen(1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994)Immunomethods 4:273.

Inhibition of Cell Growth or Proliferation

In another aspect, the invention pertains to methods of inhibiting cellgrowth. For example, the invention provides a method of inhibitinggrowth of a tumor cell in which cellular proliferation is associatedwith a meiotic kinesin, comprising contacting the tumor cell with anagent which inhibits a meiotic kinesin such that growth of the tumorcell is inhibited. Preferably, the tumor cell comprises supernumerarycentrosomes. Preferably, the meiotic kinesin is a kinesin-14 familymember, most preferably HSET. The agent can be, for example, an RNAiagent, an antisense molecule or a small molecule, as described infurther detail above. In one embodiment, the agent inhibits the ATPaseactivity of the kinesin. In another embodiment, the agent inhibits themicrotubule binding activity of the kinesin.

The tumor cell can be contacted with the agent by, for example,culturing the tumor cell with the agent. Additionally or alternatively,the tumor cell can be contacted with the agent by directly injecting theagent into a tumor that contains the tumor cell,. Additionally oralternatively, the tumor cell can be contacted with the agent byadministering the agent to a subject bearing a tumor that contains thetumor cell.

In another aspect, the invention provides a method of inhibitingproliferation of a cell in which cellular proliferation is associatedwith a meiotic kinesin, comprising contacting the cell with an agentwhich inhibits a meiotic kinesin such that inhibition of cellproliferation is achieved. Preferably, the cell contains supernumerarycentrosomes. Preferably, the meiotic kinesin is a kinesin-14 familymember, most preferably HSET. The agent can be, for example, an RNAiagent, an antisense molecule or a small molecule, as described infurther detail above. In one embodiment, the agent inhibits the ATPaseactivity of the kinesin. In another embodiment, the agent inhibits themicrotubule binding activity of the kinesin.

The cell can be contacted with the agent by, for example, culturing thecell with the agent. Additionally or alternatively, the cell can becontacted with the agent by directly injecting the agent into a tumorthat contains the cell. Additionally or alternatively, the cell can becontacted with the agent by administering the agent to a subject bearingthe cell.

Screening Methods

In another aspect, the invention pertains to methods of identifyingcompounds that inhibit the activity of a meiotic kinesin, such as HSET.For example, the invention provides a method for identifying a compoundthat inhibits activity of a meiotic kinesin HSET, the method comprising

providing an indicator composition comprising the meiotic kinesin HSET;

contacting the indicator composition with a test compound; and

determining activity of the meiotic kinesin HSET in the presence of thetest compound, wherein reduction of activity of the meiotic kinesin HSETin the presence of the test compound, as compared to activity of themeiotic kinesin MET in the absence of the test compound, identifies thetest compound as a compound that inhibits the activity of a meiotickinesin HSET.

The indicator composition can be, for example, a cell that contains(i.e., expresses) HSET or a cell-free composition that contains HSET. Ifthe indicator composition is a cell that contains (i.e., expresses)HSET, it can be a cell that naturally expresses HSET or a cell that hasbeen engineered (e.g., by standard recombinant DNA techniques) toexpress or overexpress HSET. Cells that naturally express HSET include,but are not limited to, the cancer cell lines MDA-231, MMEDX 4N andNIE-115, as well as other cell lines described in the Examples. Toengineer a cell line to express human HSET, the human HSET cDNA (havingthe sequence set forth in Genbank Accession No. NM_(—)002263) can beobtained by standard methods (e.g., PCR amplification), inserted into anexpression vector and transfected into a host cell, using standardrecombinant DNA techniques. To obtain HSET in a cell-free composition,HSET can be purified using methods known in the art. For example,DeLuca, J. G. et al. (2001) J. Biol. Chem. 276:28014-28021 describe thepurification of human HSET from HeLa cells. Anti-HSET antibodies havebeen described in the art which can be used to purify HSET by standardimmunoaffinity techniques.

The “contacting,” step of the method can comprise, for example,incubation of the test compound with a cell that contains (i.e.,expresses) HSET or incubation of the test compound with a cell-freecomposition that contains HSET.

The step of “determining activity of the meiotic kinesin HSET” can becarried out using one or more of a variety of possible “read-outs.” Forexample, in one embodiment, the activity of HSET is determined bymeasuring HSET mRNA levels in a cell that expresses HSET, wherein a testcompound is an inhibitor of HSET activity if it reduces the level ofexpression of HSET mRNA as compared to the level in the absence of thetest compounds. Methodologies for measuring HSET mRNA levels are wellestablished in the art, including but not limited to Northern blotanalysis and PCR amplification methods. In another embodiment, theactivity of HSET is determined by measuring HSET protein levels in acell that expresses HSET, wherein a test compound is an inhibitor ofHSET activity if it reduces the level of expression of HSET protein ascompared to the level in the absence of the test compounds.Methodologies for measuring HSET protein levels are well established inthe art, including but not limited to Western blot analysis andimmunoprecipitation methods.

In yet other embodiments, the activity of HSET is determined bymeasuring one or more enzymatic or biological activities of HSET. Forexample, purified HSET has been demonstrated to produce microtubulegliding when Taxol-stabilized microtubules are contacted with purifiedHSET (see DeLuca, J. G. et al. (2001) J. Biol. Chem. 276:28014-28021).Thus, in one embodiment, the effect of a test compound on HSET-mediatedmicrotubule gliding in vivo can be determined, as compared toHSET-mediated microtubule gliding in the absence of the test compound,to thereby determine the effect of the test compound on HSET activity.

Additionally or alternatively, the effect of a test compound on HSETATPase activity can be determined. For example, DeBonis, S. et al.(2004) Mol. Cancer Ther. 3:1079-1090 describe a microtubule-activatedATPase assay used to identify inhibitors of the mitotic kinesin Eg5.Similarly, such a microtubule-activated ATPase assay can be used withpurified HSET to determine the effect of a test compound on the ATPaseactivity of HSET. Thus, in one embodiment, the activity of the meiotickinesin HSET is determined by measuring the ATPase activity of thekinesin.

Other biological activities of HSET that can be used as “read-outs” indetermining the effect of a test con pound on HSET activity include, butare not limited to, microtubule binding activity and centrosomeclustering activity. Thus, in other embodiments, the activity of themeiotic kinesin HSET is determined by measuring the microtubule bindingactivity or centrosome clustering activity of the kinesin.

It has been discovered that overexpression of the HSET gene in cellsleads to growth arrest and cell death. Accordingly, the invention alsoprovides cell-based assays to determine the effect of a test compoundupon HSET-induced growth arrest and cell death, and thereby identifymodulators (e.g., inhibitors) of HSET. Generally, these assays includethe steps of: (a) providing an indicator cell comprising as recombinantexpression vector comprising an HSET gene operably linked to a promoter,under conditions whereby said HSET gene is expressed in said indicatorcell; (b) contacting said indicator cell with a test compound, and (c)determining effect of the test compound on HSET-induced growth arrestand cell death, wherein attenuation of HSET-induced growth arrest andcell death in the presence of the test compound, as compared toHSET-induced growth arrest and cell death in the absence of the testcompound, identities the test compound as a compound that inhibits theactivity of the meiotic kinesin, HSET. In one embodiment, the promoteris an inducible promoter. Any art recognized inducible promoter systemis considered, including but not limited to, an ecdysone induciblesystem (see, for example, Wakita et al., 2001, Biotechniques 31:414).Methodologies for measuring cell proliferation and cell viability arewell known in the art (see, for example, Wilson, A. P., Cytotoxicity andViability Assays in Animal Cell Culture: A Practical Approach, 3rd ed.(ed. Masters, J. R. W.) Oxford University Press: oXford 2000, Vol. 1;and Mosmann, T., Rapid Colorimetric Assay for Cellular Growth andSurvival: Application to Proliferation and Cytotoxicity Assays. J.Immunol. Meth. 1983, 65, 55-63.).

In a preferred embodiment, a library of compounds is screened in thescreening assay of the invention to identify compounds within thelibrary that exhibit the ability to inhibit the activity of HSET. Thus,in a preferred embodiment, the indicator composition described above iscontacted with each member of a library of test compounds and one ormore test compounds within the library are selected that inhibit theactivity of the meiotic kinesin HSET.

In yet another aspect, the invention pertains to isolated compoundsidentified by the screening methods of the invention.

Methods of Selecting Subjects

In another aspect, the invention pertains to methods of selectingsubjects for treatment with an agent that inhibits a meiotic kinesin,such as HSET. As demonstrated herein, inhibition of HSET activityselectively reduces the viability of cells, such as cancer cells, thatcontain extra centrosomes. Accordingly, subjects bearing is tumorcontaining cells with extra centrosomes are of particular interest fortreatment with an agent that inhibits a meiotic kinesin, such as HSET.Thus, in another aspect, the invention provides a method of selecting asubject with as tumor for treatment with an agent which inhibits ameiotic kinesin, the method comprising (i) obtaining a tumor cell samplefrom the subject, and (ii) determining centrosome number in the tumorcell sample, wherein presence of supernumerary centrosomes in the tumorcell sample selects the subject for treatment with an agent whichinhibits a meiotic kinesin. Methods for determining centrosome numbersin cells are well known in the art and can be applied to this subjectselection method. For example, centrosomes can be fluorescently stainedusing an anti-gamma tubulin antibody and a fluorescently-labeledsecondary antibody, followed by immunofluorescence imaging to quantitatethe number of centrosomes per cell (described further in the Examples).

Preferably, the meiotic kinesin is a kinesin-14 family member, morepreferably HSET. Preferably, at least 50% of tumor cells in the tumorcell sample contain supernumerary centrosomes. More preferably, at least75% of tumor cells in the tumor cell sample contain supernumerarycentrosomes. Even more preferably, at least 90% of tumor cells in thetumor cell sample contain supernumerary centrosomes.

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents of allreferences, Genbank entries, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference in their entirety.

EXAMPLE 1 Identification of therapeutic Targets for the Treatment ofMeiotic Kinesin Associated Diseases or Disorders

In this example, an RNAi screen was used to comprehensively definemolecular pathways required for clustering supernumerary centrosomes. Of8 Drosophila cell lines characterized, the near-tetraploid S2 cells weremost suitable for the screen because >50% of cells contain extracentrosomes which are efficiently clustered into two poles duringmitoses. The scheme for the genome-wide screening is illustrated in FIG.1 which provides details of the procedures for the primary and secondaryscreens.

23,172 dsRNAs targeting ˜99% of the Drosophila genome (˜14,000 genes)were screened to identify genes whose knockdown leads to multipolarspindles (centrosome de-clustering) in S2 cells. S2 cells were exposedto dsRNA for 4 days and mitotic figures were enriched by treatment withthe proteasome inhibitor MG132 during the last 9 hours of the RNAitreatment. Cells were stained for DNA, microtubules (MTs) andcentrosomes, and images were acquired with a 20× objective, using a highthroughput automated microscope.

More specifically, S2 cells were plated at a density of 1×10⁴ cells/wellin serum-free Schneider's medium in 384 well plates which werepre-plated with 0.25 μg dsRNA (dsRNAs are available at the DrosophilaRNAi Screening Center, DRSC, http://flyrnai.org). Cells were incubatedwith dsRNA for 40 minutes at room temperature (RT) in serum free medium,followed by addition of serum-containing medium and incubated for 3.5days to allow for protein depletion. To block the metaphase-anaphasetransition, 25 μM MG132, a proteosome inhibitor, was added at the end ofthe RNAi treatment (3.5 day-RNAi treated cells) and incubated for anadditional 9 hours (total of approximately 4 day RNAi incubation). Tofacilitate the attachment of mitotic cells, RNAi treated cells wereresuspended, transferred to new 384 well plates that were pre-coatedwith Concanavalin A (Con-A, 0.25 mg/ml), and the plates were spun at1,000 rpm for 1 minute. Cells were fixed in 4% paraformaldebyde (PFA) inPBS (pH 7.2), permeabilized with PBS-Triton 0.01% (PBST), incubated with0.5% SDS in PBST, and kept in FBS 4° C. until proceeding toimmunostaining.

For the primary screen, the fixed cells were stained for MTs andcentrosomes with FITC-anti-alpha tubulin (DMIA, 1:300, Sigma) and mouseanti-gamma tubulin (GTU88, 1:500) antibodies, respectively. Alexa Fluor568 or 594 Donkey anti-mouse IgG was used as secondary antibodies(1:1000). Cells were stained for DNA with Hoechst 33342 (1:5000,Invitrogen) in PBST and stored in the same solution at 4° C.

For the primary screen, cells were imaged using an automated microscope,either the ImageXpress Micro (Molecular Devices, ICCB, inverted fullyautomated epifluorescent microscope, laser auto-focus, equipped with thePhotometrics CoolSNAP ES digital CCD camera, MetaXpress for analysis),or the Discovery-1 (Molecular Devices, DRSC, automated filter anddichroic wheels and six objective turret, high-speed laser auto-focus,and can measure up to eight fluorophores per assay in multi-wellplates), using a 20× air objective. Auto-focusing; was performed on FITC(MTs) and images were acquired from single focal plane for threechannels (Hoechst, Cy3, and FITC).

The secondary screen was performed in 96 well plates (1 μg dsRNA/wellfor 5×10⁴ cells/well) and followed almost the same methodology as theprimary screen. At the end of RNAi, cells were transferred to 96 wellglass-bottom plates (Whatman) for high resolution imaging. Cells werestained additionally to identify mitotic cells with anti-rabbitphospho-histone H3 and Alexa Fluor 660 Donkey anti-rabbit IgG. To ensureimaging of all centrosomes, 3D images scare taken with a Zeiss Axiovertmicroscope and Slidebrook software (Intelligent Imaging Inovations,Denver, Colo.) using a 40× air ELWD objective (Zeiss) with 1 μm stepsize. The height (start and end point) of Z stacks were manuallyadjusted for all 701 RNAi conditions:

By visual inspection of ˜96,000 images, the percentage of multipolarspindles for each RNAi condition was scored. The screening results aresummarized below in Table 1.

TABLE 1 Summary of RNAi Screen Results in S2 Cells Number Total Screened23,172 dsRNAs Not determined (# spindle <10) 148 Scored as hits inprimary screen 701 genes Tested hits from primary screen 292 genes Totalhits after secondary screen 133 (46%) genes Hits with mammalian homologs82 (62%) genesUsing a 95% confidence interval, the primary screen identified 701candidates associated with a multipolar spindle phenotype. 292 geneswere selected as in cohort for further study based on: the strength ofthe phenotype, the existence of readily identifiable mammalianhomologues, and few or no predicted off-target effects. Additionally,most genes that were previously determined to be required forcytokinesis in Drosophila cells were eliminated (Echard, A. et al.(2004) Curr Biol 14:1685-1693; Eggert, U.S. et al. (2004) PloS Biol2;e379) because spindle multipolarity can be a secondary effect ofcytokinesis failure (Goshima, G. et al. (2007) Science 316:417-421). Ofthe 292 genes selected for the secondary screen, 133 were confirmed tohave a bona fide role in centrosome clustering. Among the validatedgenes, 62% of the genes identified (83 out of 133 genes) have mammalianhomologues, while 33% of the genes (44) do not have a known function.Centrosome clustering can occur with varying efficiency. The followingclasses of defects were distinguished; bipolar spindles with multiplecentrosomes scattered around the spindle, small multiaster spindles andlarge multipolar spindles.

The screen identified genes involved in a diverse range of cellularprocesses, suggesting unappreciated complexity in the mechanismscontrolling organization of supernumerary centrosomes, including anumber of genes that promote the bundling of spindle MTs, for example,the minus end-directed kinesin Ncd (human HSET). The screen alsoidentified genes in unexpected processes. The discovery of genesrequired for the SAC, actin, cell polarity and cell adhesion suggestednovel mechanisms that suppress multipolar mitoses. Below are presentedexperiments that define three overlapping mechanisms that suppressmultipolar mitoses: a timing mechanism employing the SAC, intrinsic poleclustering mechanisms relying on MT regulators, and a novel mechanismrequiring actin and cell adhesion.

EXAMPLE 2 The Spindle Assembly Checkpoint (SAC) Prevents MultipolarMitoses

The SAC components Mad2 BubR1 (human Bub1) and CENP-Meta (human CENP-E)are required for centrosome clustering. FIG. 2 illustrates that Mad2 isrequired for centrosome clustering. Centrosome clustering defects werescored in S2 cells upon RNAi of EGFP, Mad2 alone and EGFP or Mad2 plus 7hours of MG132 treatment. The graph of FIG. 2 shows the average of threeindependent experiments (mean±Sd, *p<0.05; ***p<0.001, Student's ttest).

The results shown in FIG. 2 indicate a role for the SAC in the processof centrosome clustering. This requirement was even more evident incells that were not treated with MG132, indicating that the shorttreatment with MG132 employed in the screen partially masked the effectof SAC gene RNAi on spindle multipolarity. This finding was somewhatsurprising, given previous work in PtK1 cells suggesting that the SAC isnot activated by multipolar spindles or multiple centrosomes (Sluder. G.et al. (1997) J Cell Sci 110 (Pt 4): 421-429).

Time-lapse imaging supported a role for the SAC in preventing multipolarmitoses. In S2 cells where centrioles and MTs were labeled withGFP-SAS-6 and mCherry α-tubulin, there was a clear correlation betweenan increased number of centrosomes and a prolonged time required to forma bipolar spindle (2.7 fold). The interval between NEBD and anaphaseonset was measured (visualized with GFP-Cid, Drosophila CENP-A)comparing cells with 2 or >2 centrosomes. Relative to cells with 2centrosomes, cells with multiple centrosomes exhibited a marked delay inanaphase onset (1.8 fold). Moreover, the delay in anaphase onset wasabolished by Mad2 RNAi, and these cells entered anaphase withdeclustered centrosomes and misaligned kinetochores. Further suggestingSAC activation, multipolar preanaphase spindles had a strong increase inthe number of BubR1 foci relative to bipolar metaphase spindles.Finally, the requirement for the SAC to prevent multipolar mitoses canbe partially suppressed by an artificial metaphase delay imposed bytreatment with MG132. This suggests that the SAC does not monitormultipolar mitosis per se but rather that SAC activation, likelytriggered by abnormal kinetochore attachment or tension, providessufficient time for compensatory mechanisms to organize multiplecentrosomes.

EXAMPLE 3 Spindle-Intrinsic Pole Clustering Forces Prevent MultipolarMitoses

Previous work in S2 cells has demonstrated a critical role for MT motorsand MAPs in spindle pole focusing (Goshima, G. et al. (2005) J Cell Biol171:229-240; Morales-Mulia, S. and Scholey, J. M. (2005) Mol Biol Cell16:3176-3186). The screen described in Example 2 1 identified Ncd, aKinesin-14 family member, as the strongest hit in the primary screen.Ncd is a minus end-directed motor that bundles MTs at the spindle poles(Karabay, A. and Walker, R. A. (1999) Biochemistry 38:1838-1849). ByGFP-SAS-6 labeling, it was demonstrated that Ncd is required to clustermultiple centrosomes. Drosophila dynein was not identified in thescreen. This is expected because in S2 cells loss of dynein does notsignificantly induce multipolar mitoses, although it does compromisecentrosome attachment and tight focusing of the spindle poles (Goshima,G. et al. (2005) J Cell Biol 171:229-240). Further validation of thescreen confirmed the role of the MAP Asp in pole focusing(Morales-Mulia, S. and Scholey, J. M. (2005) Mol Biol Cell 16:3176-3186;Wakefield, J. G. et al. (2001) J Cell Biol 153:637-648).

Additionally, the screen identified several other factors thatcontribute to the intrinsic cohesion of spindle MTs. A requirement forBj1/RCC1 (RanGEF) in centrosome clustering was identified, consistentwith its role in preventing multipolar mitosis in mammalian cells (Chen,T. et al. (2007) Nat Cell Biol 9:596-603). Roles for theADP-ribosylation factors Tankyrase and CG15925, a putative human PARP-16homolog were also identified (Schreiber, V. et al. (2006) Nat Rev MolCell Biol 7:517-528). ADP-ribosylation by tankyrase is thought tocontribute to spindle bipolarity by providing a static matrix that mayanchor MT motors and other spindle proteins (Chang, P. et al. (2005) NatCell Biol 7:1133-1139). A role for PARP-16 in mitosis has not beenpreviously described.

EXAMPLE 4 Actin-Dependent Forces Regulate Spindle Multipolarity

In addition to genes that likely contribute to the bundling andorganization of spindle MTs, unexpectedly, genes involved in theorganization and regulation of the actin cytoskeleton, such as theformin Form3/INF2 were also identified (Chhabra. E. S. and Higgs, H. N.(2006) J Biol Chem 281:26754-26767). Knockdown of these genes does notinduce multipolar mitoses indirectly by triggering cytokinesis failure.Experiments using a brief (2 hr) treatment with small molecules thatdisrupt the actin cytoskeleton similarly induce multipolar mitoses.

More specifically, cells were treated with Latrunculin (40 μM LatA).Cytochalasin D (20 μM) for 2 hours and the percentage of centrosomeclustering defects was determined. The results are shown in FIG. 3,which demonstrates the requirement for the actin cytoskeleton forcentrosome clustering in S2 cells. The graph shows the average of threeindependent experiments (mean±SD, *p<0.05; **p<0.005, Student's t test).

Furthermore, live cell imaging of S2 cells revealed that actin is indeedrequired for the initial clustering of multiple centrosomes. Relative tocontrols (14.7±6.4 min), there was a 1.5 fold delay in centrosomeclustering in 13/15 LatA-treated cells (27.1±12.3 min). The remaining2/15 cells completely failed to cluster extra centrosomes. The cellcycle delay induced by LatA treatment is likely due to activation of theSAC, as evidenced by prominent labeling of kinetochores with BubR1 mLatA-treated cells.

Next, it was determined if cortical contraction is required forcentrosome clustering. Cells were exposed, for 2 hours, to solubletetravalent lectin concanavalin A (Con-A), which crosslinks the plasmamembrane and thus globally blocks cortical contraction (Canman, J. C.and Bement, W. M. (1997) J Cell Sci 110 (Pt 16):1907-1917). Thepercentage of centrosome clustering defects after Con-A treatment wasdetermined. The results also are shown in FIG. 3, which demonstratesthat Con-A treatment induced centrosome clustering defects.

Furthermore, it was found that enhancing myosin-based contractility cansuppress spindle multipolarity. Low concentrations of calyculin A (CA)inhibit the myosin light chain phosphatase (MLCP) and promote myosin IIactivation without altering its distribution (Gupton, S. M. andWaterman-Storer, C. M. (2006) Cell 125:1361-1374). Wild type S2 cellstreated with CA had a modest decrease in centrosome clustering defects(15% to 9%). Moreover, CA treatment partially rescued the centrosomeclustering defect induced by Ncd RNAi. Thus, in cells with extracentrosomes, actin and actin-based contractility influence whethermitosis is bipolar or multipolar.

It was also determined whether the actin and intrinsic spindle forcesact cooperatively to prevent multipolar mitoses. Indeed, LatA treatmentin Ncd or Bj1/RCC1-depleted cells resulted in a combinatorial increasespindle multipolarity. To further evaluate this idea, time-lapsespinning disc microscopy was used to define the trajectory of centrosomemovement in Ncd-depleted S2 cells. In contrast to control cells,centrosomes in Ncd-depleted cells exhibited a striking increase inmobility; both the speed and extent of movement was increased. Moreover,the bulk of the centrosome movement was directed away from the spindleand towards the cell cortex. The mobility of centrosomes in Ncd-depletedcells was severely diminished by transient exposure of cells to LatA,demonstrating an actin requirement for these cortical pulling forces.Thus, cortical forces collaborate with intrinsic spindle bundling forcesto organize multiple centrosomes.

These results also provided insight into the nature of the corticalforce generators that regulate spindle multipolarity. It was found thatthe MT+tip CLIP-190 and the myosin Myo10A are important for centrosomeclustering. Drosophila Myo10A is a human Myo15 homolog that can bind MTsvia a unique MyTH4-FERM domain. Myo10, a member of mammalianMyTH4-FERM-containing myosin, is known to be required for spindlepositioning (Sousa, A. D. and Cheney, R. E. (2005) Trends Cell Biol15:533-539; Toyoshima, F. and Nishida, E. (2007) EMBO J 26:1487-1498;Weber, K. L. et al. (2004) Nature 431:325-329). RNAi of Myo10A but notthe other Drosophila MyTH4-FERM-containing myosin Myo7 induced a 2-foldincrease in centrosome clustering defects without cytokinesis failure(Eggert, U. S. et al. (2004) PLoS Biol 2, e379). Moreover, knockdown ofMyo10A did not have an additive effect on spindle multipolarity if cellswere concomitantly treated with LatA. Finally, centrosome tracking ofMyo10A-depleted cells revealed as similar effect on centrosome movementas LatA treatment; in cells depleted of Myo10, only random or greatlyreduced movements of centrosomes were detected in contrast to theextensive cortically-directed movement shown in Ncd depleted cells.Together, the data demonstrate that multiple centrosomes are organizedcombinatorially by spindle-intrinsic forces and by actin-dependentcortical forces acting at least in part on astral MTs.

EXAMPLE 5 Cell Shape, Cell Polarity and Adhesion Effects on SpindleMultipolarity

The screen identified a requirement for genes implicated in celladhesion for centrosome clustering: Turtle, Echinoid, Cad96Ca, CG33171,and Fit1. The Drosophila FERM domain containing protein Fit1 appears tohave a highly conserved function in regulating cell-matrix adhesion inhigher eukaryotes (Rogalski. T. M. et al. (2000) J Cell Biol150:253-264; Tu, Y. et al. (2003) Cell 113:37-47). The mammalian Fit1homolog, Mig-2/human PLEKHC1, localizes to focal adhesions (FAs) and isimportant for integrin-mediated cell adhesion and modulation of cellshape by integrins to actin cytoskeleton (Tu, Y. et al. (2003) Cell113:37-47). The uncharacterized CG33171 protein has homology tomammalian Col18A, previously implicated in the regulation of cell matrixadhesion (Dixelius, J. et al. (2002) Cancer Res 62:1944-1947; Wickstrom,S. A. et al., (2004) J Biol Chem 279:20178-20185). Turtle and Echinoidcontaining fibronectin type-III domains are involved in cell-celladhesion (Bodily, K. D. et al. (2001) J Neurosci 21:3113-3125; Wei, etal. (2005) Dev Cell 8:493-504). In addition, the posterior/lateralpolarity gene PAR-1 (PAR-1/MARK/KIN1 family member) and the apicalpolarity genes Crumbs and Cornetto were identified, which are importantfor astral MT function, asymmetric cell division and epithelial polarity(Bulgheresi, S. et al. (2001) J Cell Sci 114:3655-3662; Munro, E. M.(2006) Curr Opin Cell Biol 18:86-94; Tepass, U. et al. (2001) Annu RevGenet 35:747-784). A number of these genes have previously beenidentified because of their requirement to maintain normal interphasecell shape and adhesion. Indeed, LatA treatment or Myo10 depletionshowed no enhancement of spindle multipolarity when combined withdepletion of CG33171, Fit1, Crumbs, Cornetto, or PAR-1 proteins,demonstrating that these genes influence centrosome clustering via theactin cytoskeleton.

EXAMPLE 6 Conservation of the Mechanisms to Prevent Multipolar Mitoses

It was next determined if mammalian cancer cells utilize similarmechanisms to cluster multiple centrosomes as observed in S2 cells. Thiswas done to establish the relevance of the screen to human cancer andbecause of techniques utilizing mammalian cancer cells that enableddirect characterization of adhesion and cell shape effects on multipolarmitoses. Although there is some variability in its efficiency,clustering of extra centrosomes is commonly observed in mammalian cells.

To determine the effect of transient actin disruption in mammaliancells, the cell lines MCF-7, MDA-231, MMEDX 4N and NIE-115 were treatedwith DMSO, LatA (5 μM) or Dihydrocytochalasin B (DCB) (10 μM) for 2hours. The MCF-7 cell line contains 2 centrosomes whereas the other celllines contain greater than 2 centrosomes. The results are shown in FIG.4. The graph shows the average of three independent experiments(mean±SD, *p<0.05; **p<0.005; ***p<0.001, Student's t test). Asillustrated in FIG. 4, transient actin disruption led to a significantincrease in the frequency of multipolar spindles in cell lines thatcontained multiple centrosomes but not in cells with normal centrosomenumber. These multipolar spindles result from declustering of extracentrosomes and were not due to the splitting/fragmentation ofcentrioles. Actin presumably influences centrosome positioning viaforces on astral MTs. Consistent with this idea, low dose nocodazoletreatment, selectively disassembling astral MTs (Thery, M. et al. (2005)Nat Cell Biol 7;947-953), increased the frequency of multipolar spindlesspecifically in cells with extra centrosomes.

The parallel between Drosophila and mammalian cells extended to thegenetic requirements for centrosome clustering. To examine this further,siRNA experiments were performed with mammalian HSET (Ncd homolog) andMyo10 genes, as follows. Mixed pools (ON-TARGETplus and SMART pools) of4 different oligo of siRNAs against human HSET, human Myo10 and mouseMyo10 were purchased from Dharmacon, siRNA against mouse HSET waspurchased from Ambion. Non-specific scrambled siRNA was used as control(Ambion). The oligo sequence of the siRNAs are set forth below in Table2:

TABLE 2 siRNA Oligo Sequences Oligo Sequence 5′-3′ siRNA ID# SEQ #Human HSET UAACUGACCCUUUAAGUCCUU J-004958-06  1 AGUGUUGUGCGCUCUGUCCUUJ-004958-07  2 GACACAAGCACGCAAGUUCUU J-004958-08  3UGGUCCAACGUUUGAGUCCUU J-004958-09  4 Human Myo10 CAAGUUGAGAUUUAUGUCCUUJ-007217-05  5 UAAGACAUCAGCUACGACGUU J-007217-06  6UAAUCUACAAUUCUCCCGCUU J-007217-07  7 AUUCCCUGAAAUUUCCUCCUU J-007217-08 8 Mouse HSET GGCUAAUAAGAAGUGAAGtt 287750  9 GGAACUGAAGGGCAAUAUCtt287551 10 GGCCAUUAACAGCAGUCUGtt 287752 11 Mouse Myo10UUCCACGGUGCCCUUGAGCUU J-062004-09 12 UUCUCCUCGCUAUCGUUUUUU J-062004-1013 UUUCUUGUGCAGCCAGCCUUU J-062004-11 14 UACAUCAGCUUCGACUGGCUUJ-062004-12 15

Cells were transfected with Lipofectamine RNAiMAX (Invitrogen) with afinal siRNA concentration of 50 nM according to the manufacturer'sinstructions. Cells were analyzed/harvested three days aftertransfection (unless specified otherwise). FIGS. 5 and 6 show theresults of Western blots showing the depletion of HSET (FIG. 5) or Myo10(FIG. 6) after three days of siRNA in MDA-231 and MCF-7 cells. FIG. 7 isa bar graph showing the percentage of cells with multipolar spindles inMCF-7 or MDA-231 cells upon treatment with siRNA of HSET Myo10. Theresults shown in FIG. 7 demonstrate that siRNA of the Ncd homolog HSET(a Kinesin-14 member) and Myo10 increased the frequency ofmultipolarity, specifically in cells harboring multiple centrosomes(i.e., MDA-231 cells). As in S2 cells, Myo10-induced multipolarity isnot a consequence of cytokinesis failure.

Finally, it was determined whether the cortical actin cytoskeletoninfluences centrosome organization in parallel with intrinsic spindlepole clustering forces, by treatment with siRNA of HSET or Myo10 incombination with the actin disruption agent LatA. As shown in FIG. 8,disruption of both actin and HSET had a combinatorial effect, increasingthe frequency of multipolar spindles relative to the individualtreatments. When Myo10 siRNA is combined with LatA treatment no sucheffect was observed. Thus, similar overlapping mechanisms preventmultipolar mitoses in mammalian cancer cells and Drosophila S2 cells.

EXAMPLE 7 Interphase Cell Shape, Adhesion and Multipolar Mitoses

Although cells round up in mitosis, the preserve a memory of theirinterphase shape by retaining actin-containing retraction fibers (RFs)linked to sites of strong cell-matrix adhesion (Mitchison, T. J. (1992)Cell Motil Cytoskeleton 22:135-151; Thery, M. and Bornens, M. (2006)Curr Opin Cell Biol 18:648-657). The interphase adhesion pattern and thedistribution of actin-containing RFs are known to strongly influencespindle orientation during mitosis (Thery, M. et al. (2007) Nature447:493-496; Thery, M. et al. (2005) Nat Cell Biol 7:947-953). Thefinding from the screen that preventing multipolar mitoses requires bothcell matrix adhesion genes and actin regulators, suggested an appealinghypothesis: these gene products could act cooperatively to organizeextra centrosomes by affecting the distribution and/or the compositionof retraction fibers (RFs) and thus cortical force generators.

Several lines of evidence support this hypothesis. First, live-cellimaging was used to correlate interphase cell shape with the pattern ofcell division during mitosis. MDA-231 (breast cancer cells containingextra centrosomes) that assumed an elongated or polarized shape ininterphase, almost uniformly underwent bipolar divisions. By contrast,MDA-231 cells that assumed a round shape in interphase had an increasedfrequency of multipolar divisions. Second, in tetraploid BSC-1 cells,whose thick RFs are readily visualized by DIC imaging, it was noted thata strong correlation between the positioning of the RFs and whethercells underwent a bipolar (bipolar distribution of RFs) or multipolardivision (isotrophic distribution of RFs). Third, RFs accumulatespecific proteins, such as the ERM protein ezrin, which are implicatedin cortical heterogeneity and thus local force generation on astral MTs.Disruption of this cortical heterogeneity by the src kinase inhibitorPP2 (Thery, M. et al. (2005) Nat Cell Biol 7:947-953), also inducedmultipolar spindles in MDA-231 but not in MCF-7 cells. Fourth, toevaluate the role of cell matrix adhesion for the efficiency of mitosis,cells were plated on different concentrations of fibronectin (FN) tovary the strength of cell-matrix attachment. Concentrations of FN thatinhibit focal adhesion (FA) turnover (30 μg/ml) increased the frequencyof multipolar spindles in MDA-231 cells but not in MCF-7 cells.Moreover, this effect could be reversed by CA, which promotes FAturnover by increasing cortical contractility (Gupton, S. L. andWaterman-Storer, C. M. (2006) Cell 125:1361-1374).

To directly test the role of the cell adhesion pattern and RFpositioning in centrosome clustering, micro-contact printing of FN wasused to manipulate cell adhesion patterns (Thery, M. et al. (2005) NatCell Biol 7:947-953). MDA-231 cells plated onto Y-shaped or O-shapedmicropatterns had a significant (3-4 fold) increase of multipolarspindles compared to the controls. In contrast, plating cells onH-shaped micropattern suppressed the frequency of multipolar spindlesrelative to control cells (2%, half of the control). Thus, O and Yarrangements of adhesive contacts bias cells into multipolar mitoseswhereas bipolar arrangements of adhesive contacts (H-shape) promotebipolar mitoses. These findings demonstrate that interphase celladhesion pattern, and thus cell shape, can have a remarkable influenceon the fidelity of mitosis—specifically in cancer cell containing extracentrosomes.

EXAMPLE 8 Disruption of Centrosome Clustering can Selectively KillCancer Cells

In principle, disruption or centrosome clustering could have a selectiveeffect on the viability of cancer cells containing multiple centrosomesbecause most somatic cells have two centrosomes during mitosis. As afirst step towards evaluating this potential therapeutic strategy, thesensitivity of different cancer cell lines to knockdown of HSET wasdetermined.

HSET is a particularly interesting therapeutic target because it isnon-essential for cell division in normal cells and kinesins areamenable to small molecule inhibition (Mayer, T. U. et al. (1999)Science 286:971-974; Mountain, V. et al. (1999) J Cell Biol147:351-366). Depletion of HSET by SiRNA leads to an increase inmultipolar spindles in human cancer cells containing multiplecentrosomes (see FIG. 7, described further in Example 6 above). Todetermine the consequences of centrosome de-clustering, cell divisionwas monitored in multiple cell lines that contain extra centrosomesusing DIC microscopy. Depletion of human HSET, confirmed with 3independent siRNAs, induced a dramatic increase in multipolar anaphases(88%) in NIE-115 cells in which nearly 100% of cells contain extracentrosomes (Spiegelman, B. M. et al. (1979) Cell 16:253-263). A similarresult was obtained with MDA-231 cells where 50% of cells contain extracentrosomes (24% after HSET depiction) and with tetraploid BJ andNIH-3T3 cells with extra centrosomes. By contrast, HSET knockdown had noeffect on cell division in a variety of diploid control cells.

FIG. 9 illustrates the loss of cell viability and inhibition of colonyformation by NIE-115 cells after 6 days of HSET siRNA. FIG. 9 showsrelative cell number in control and HSET-depleted (−HSET) cells fromthree independent experiments after six days post transfection (leftpanel) and the average colony number from two independent experiments in4 different areas (right panel area=10 mm²). Strikingly, depletion ofHSET from NIE-115 cells for 6 days, by siRNA treatment, reduced cellviability more than 90%, and many of the surviving cells were shown tobe senescent.

FIG. 10 illustrates HSET RNAi-induced cell death in various cancer celllines in proportion to the fraction of cells with extra centrosomes. Thebar graph shows relative cell number in control and HSET-depleted(−HSET) cells after six days post-transfection with siRNA. Thepercentage of cells with greater than 2 centrosomes is indicated belowin the graph. The graph shows the average of three independentexperiments. All graphics represent mean±SD (**p<0.005, ***p<0.001,Student's t test). Thus, HSET depletion induced cell death in variouscancer cells lines in rough proportion to the fraction of cellscontaining extra centrosomes. In contrast, the viability of cells thatmostly possess two centrosomes was only slightly reduced in the absenceof HSET. Thus, centrosome de-clustering can induce cell deathselectively in cells with supernumerary centrosomes.

1-36. (canceled)
 37. A method of selecting a subject with a tumor fortreatment with an agent which inhibits a meiotic kinesin selected from akinesin-14 family member, the method comprising (i) obtaining a tumorcell sample from the subject, and (ii) determining centrosome number inthe tumor cell sample, wherein the presence of supernumerary centrosomesin the tumor sample selects the subject for treatment with an agentwhich inhibits the meiotic kinesin.
 38. (canceled)
 39. The method ofclaim 37, wherein the meiotic kinesin is HSET.
 40. The method of claim37, wherein at least 50% of tumor cells in the tumor cell sample containsupernumerary centrosomes.
 41. The method of claim 37, wherein at least75% of tumor cells in the tumor cell sample contain supernumerarycentrosomes.
 42. The method of claim 37, wherein at least 90% of tumorcells in the tumor cell sample contain supernumerary centrosomes. 43.The method of claim 37, wherein the agent is selected from the groupsconsisting of RNAi agent, an antisense agent, and a small molecule. 44.The method of claim 37, wherein the determining step comprisesimmunofluorescence imaging to quantitate the number of centrosomes percell.